IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4

Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.     

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.     

IL-21 induces apoptosis of antigen-specific CD8+ T lymphocytes

IL-21, a member of the common gamma-chain family of cytokines, has pleiotropic effects on T, B, and NK cells. We found that IL-21 and the prototype common gamma-chain cytokine IL-2 can stimulate proliferation and cytokine secretion by Ag-specific rhesus monkey CD8+ T cells. However, unique among the members of this family of cytokines, we found that IL-21 drives these cells to apoptosis by down-regulation of Bcl-2. These findings suggest that IL-21 may play an important role in the contraction of CD8+ T cell responses.     

Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection

IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.     

Intestinal fibroblast-derived IL-10 increases survival of mucosal T cells by inhibiting growth factor deprivation- and Fas-mediated apoptosis

Mucosal T cells are essential to immune tolerance in the intestine, an organ constantly exposed to large amounts of dietary and bacterial Ags. We investigated whether local fibroblasts affect mucosal T cell survival, which is critical for maintenance of immune tolerance. Coculture with autologous fibroblasts significantly increased viability of mucosal T cells by inhibiting IL-2 deprivation- and Fas-mediated apoptosis, an effect that was both contact- and secreted product-dependent. Investigation of anti-apoptotic factors in the fibroblast-conditioned medium (FCM) revealed the presence of IL-10 and PGE2, but not IFN-beta, IL-2, or IL-15. Although recombinant IFN-beta, but not PGE2, effectively prevented T cell apoptosis, neutralizing Ab studies showed that only IL-10 blockade significantly increased T cells apoptosis, whereas neutralizing IFN-beta or IFN-alpha failed to inhibit the anti-apoptotic effect of FCM. To confirm that fibroblast-derived IL-10 was responsible for preserving mucosal T cell viability, IL-10 mRNA was demonstrated in fibroblasts by Southern blotting and RT-PCR. When FCM was submitted to HPLC fractionation, only the peak matching rIL-10 contained the anti-apoptotic activity, and this was eliminated by treatment with an IL-10-neutralizing Ab. Finally, when fibroblasts were transiently transfected with IL-10 antisense oligonucleotides, the conditioned medium lost its T cell anti-apoptotic effect, whereas medium from fibroblasts transfected with IFN-beta antisense oligonucleotides displayed the same anti-apoptotic activity of medium from untransfected fibroblasts. These results indicate that local fibroblast-derived IL-10 is critically involved in the survival of mucosal T cells, underscoring the crucial importance of studying organ-specific cells and products to define the mechanisms of immune homeostasis in specialized tissue microenvironments like the intestinal mucosa.     

MAPK/ERK signaling in activated T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly

When T cells are activated, the expression of the CD95 ligand is elevated, with the purpose of inducing apoptosis in target cells and to later eliminate the activated T cells. We have shown previously that mitogen-activated protein kinase (MAPK or ERK) signaling suppresses CD95-mediated apoptosis in different cellular systems. In this study we examined whether MAPK signaling controls the persistence and CD95-mediated termination of an immune response in activated T cells. Our results show that activation of Jurkat T cells through the T cell receptor immediately suppresses CD95-mediated apoptosis, and that this suppression is mediated by MAPK activation. During the phase of elevated MAPK activity, the activation of caspase-8 and Bid is inhibited, whereas the assembly of a functional death-inducing signaling complex (DISC) is not affected. These results explain the resistance to CD95 responses observed during the early phase of T cell activation and suggest that MAPK-activation deflects DISC signaling from activating caspase-8 and Bid. The physiological relevance of the results was confirmed in activated primary peripheral T cells, in which inhibition of MAPK signaling markedly sensitized the cells to CD95-mediated apoptosis.     

CD8 T cells inhibit IgE via dendritic cell IL-12 induction that promotes Th1 T cell counter-regulation.

Th1 and Th2 cells are counterinhibitory; their balance determines allergic sensitization. We show here that CD8 T cell subsets break these rules as both T cytotoxic (Tc)1 and Tc2 cells promote Th1 over Th2 immunity. Using IL-12(-/-), IFN-gamma(-/-), and OVA(257-264)-specific Valpha2Vbeta5 TCR-transgenic mice, we have identified the key steps involved. OVA-specific IFN-gamma(-/-) CD8 T cells inhibited IgE responses equivalent to wild-type CD8 T cells (up to 98% suppression), indicating that CD8 T cell-derived IFN-gamma was not required. However, OVA-specific CD8 T cells could not inhibit IgE in IFN-gamma(-/-) recipients unless reconstituted with naive, wild-type CD4 T cells, suggesting that CD4 T cell-derived IFN-gamma did play a role. Transfer of either Tc1 or Tc2 Valpha2Vbeta5 TCR-transgenic CD8 T cells inhibited IgE and OVA-specific Th2 cells while promoting OVA-specific Th1 cell responses, suggesting a potential role for a type 1 inducing cytokine such as IL-12. CD8 T cells were shown to induce IL-12 in OVA(257-264)-pulsed dendritic cells (DC) in vitro. Furthermore, CD8 T cells were unable to inhibit IgE responses in IL-12(-/-) recipients without the addition of naive, wild-type DC, thus demonstrating a pivotal role for IL-12 in this mechanism. These data reveal a mechanism of IgE regulation in which CD8 T cells induce DC IL-12 by an IFN-gamma-independent process that subsequently induces Th1 and inhibits Th2 cells. Th1 cell IFN-gamma is the final step that inhibits B cell IgE class switching. This demonstrates a novel regulatory network through which CD8 T cells inhibit allergic sensitization.     

Stabilized beta-catenin potentiates Fas-mediated T cell apoptosis

In response to Ag stimulation, Ag-specific T cells proliferate and accumulate in the peripheral lymphoid tissues. To avoid excessive T cell accumulation, the immune system has developed mechanisms to delete clonally expanded T cells. Fas/FasL-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) T cells. Using transgenic mice expressing a stabilized beta-catenin (beta-cat(Tg)), we show here that beta-catenin was able to enhance apoptosis of activated T cells by up-regulating Fas. In response to staphylococcal enterotoxin B stimulation, beta-cat(Tg) mice exhibited accelerated deletion of CD4(+)Vbeta8(+) T cells compared with wild type mice. Surface Fas levels were significantly higher on activated T cells obtained from beta-cat(Tg) mice than that from wild type mice. Additionally, T cells from beta-cat(Tg) mice were more sensitive to apoptosis induced by crosslinking Fas, activation-induced cell death, and to apoptosis induced by cytokine withdrawal. Lastly, beta-catenin bound to and stimulated the Fas promoter. Therefore, our data demonstrated that the beta-catenin pathway was able to promote the apoptosis of activated T cells in part via up-regulation of Fas.     

IL-12 p35 silenced dendritic cells modulate immune responses by blocking IL-12 signaling through JAK-STAT pathway in T lymphocytes

Dendritic cells (DC) constitute a complex system of uniquely specialized antigen-presenting cells (APC) that play crucial roles in the initiation and regulation of immune responses. Recent studies have demonstrated that DC silenced by siRNA IL-12 p35 showed tolerogenic capacity in vitro. However, their mechanism of action is not fully understood. In this study, IL-12p35 siRNA was chemically synthesized and transfected into DCs. A coculture of T cells and DCs was performed. After 30 min coculture, T cells were harvested and analyzed. We showed that the IL-12 p35 silenced DCs decreased IL-12-induced T cell responses through blocking tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4 proteins in T cells. These results demonstrate IL-12 p35 silenced DCs modulate immune responses by blocking IL-12 signaling through JAK-STAT pathway in T cells.     

Fas-mediated apoptosis causes elimination of virus-specific cytotoxic T cells in the virus-infected liver

Immunity to allogeneic MHC Ags is weak in rodent livers, raising questions as to the mechanisms that might control responses in this organ. Infection with an adenovirus vector reveals that T cell-mediated immunity to nonself-Ags in the liver is self-limiting. Virus-induced liver injury decreases and coincides with disappearance of virus-specific CTL, concomitant to an increase of apoptotic T cells early after infection. But whereas death in CD4 cells is independent of Fas, perforin, and TNF-alpha, that of CD8 cells requires Fas and not perforin or TNF-alpha pathways. Fas ligand is expressed on liver-infiltrating cells, pointing to death by fratricide that causes almost complete disappearance of virus-specific CTL 4 wk after infection. CTL elimination is virus dose dependent, and high doses induced high alanine aminotransferase values, elevated expression of Fas ligand on CD8 cells, and increased CD8 cell migration into the infected liver.     

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.     

TCR-independent activation of extrathymically developed, self antigen-specific T cells by IL-2/IL-15

Naive intrathymically developed T cells, which express foreign Ag-specific TCR, do not express IL-2R. After antigenic stimulation, they express high affinity IL-2R, which enables IL-2 to be used as an autocrine growth factor. On the contrary, extrathymically developed T cells, which express self Ag-specific TCR but are unresponsive to antigenic stimulation, spontaneously express low affinity IL-2R. In this study, we compared the responses of these two subsets of T cells to IL-2R stimulation and examined the influences of TCR-mediated signaling on the responses. IL-2 or IL-15 augmented the proliferative response of Ag-stimulated, intrathymically developed T cells. On the other hand, extrathymically developed T cells proliferated in response to IL-2 or IL-15, independently of Ag stimulation. Furthermore, both IL-2 and IL-15 induced IFN-gamma production of these T cells, which is strikingly augmented by the presence of IL-12. These results revealed functional differences between intrathymically developed, foreign Ag-specific T cells and extrathymically developed, self Ag-specific T cells. The latter can be activated by some inflammatory cytokines, in an Ag-independent manner, similar to NK cells.     

Rapid signaling to B cells by antigen-specific T cells requires CD18/CD54 interaction.

This study reports early B and T cell signaling events during cognate interactions between a human B cell line pulsed with peptide and an Ag-specific T cell clone. As has been previously reported, peptide in the context of the appropriate class II molecule stimulated a rise in intracellular calcium [Ca2+]i in the Ag-specific T cell clone. The activation of the T cell clone was associated with a reciprocal rise in [Ca2+]i in the B cells. Engagement of receptors on the B cell surface by the T cell also was associated with inositol phospholipid turnover comparable to that elicited by stimulation through sIg. Early signaling events in B cells can therefore be stimulated in cognate interactions with Ag-specific T cells, without the direct engagement of Ig receptors. A class II deficient B lymphoblastoid mutant, 6.1.6, which was incapable of presenting peptide to the T cell clone, could be stimulated to produce a rise in [Ca2+]i if the T cell clone was activated by monoclonal antibodies to CD3. Therefore, the interaction of class II molecules on the B cell with the TCR and/or the CD4 accessory molecule was not essential for T-dependent B cell activation. However, T-dependent signalling of B cells was profoundly inhibited by mAb to CD18 (beta-chain of LFA-1) on the T cell or CD54 (ICAM-1) on the B cell, demonstrating the importance of this pair of adhesion molecules in early T-B cell interactions.     

B cells as antigen-presenting cells: antigen-specific IL-2 production by cloned T cells without expression of IL-2 receptors

A murine T cell clone, 24-2C, responds specifically to human IgG (HGG) in the context of I-Ab. B cells purified from mouse spleen cells were examined for their function as antigen-presenting cells (APC) in the response of 24-2C cells to HGG. B cells functioned as APC for IL-2 production but not for proliferation, whereas spleen cells or spleen-adherent cells functioned as APC for both IL-2 production and proliferation. LPS-activated B cells also failed to induce the proliferative response. The addition of the culture supernatant of 24-2C cells stimulated with HGG presented by irradiated spleen cells to the culture of 24-2C cells, irradiated B cells, and HGG induced the proliferative response of 24-2C cells, whereas IL-1, IL-3, and/or interferon-gamma did not reconstitute the proliferation. The expression of IL-2 receptors (IL-2R) on 24-2C cells was examined using a monoclonal anti-mouse IL-2R antibody AMT 13 or 7D4. 24-2C cells cultured with spleen cells as APC expressed IL-2R. Those cultured alone or with B cells as APC did not express IL-2R. Enlargement of 24-2C cells in response to HGG was also examined, and the relative cell size of those cultured with B cells or spleen cells as APC was larger than that of those cultured alone. These results demonstrate that B cells as APC induce IL-2 production and cell size enlargement in the response of 24-2C cloned T cells to HGG, but not IL-2R expression nor proliferation.     

Inflammatory cytokine regulation of Fas-mediated apoptosis in thyroid follicular cells.

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of gamma-interferon and either tumor necrosis factor-alpha or interleukin 1beta allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.     

IL-12R beta 2 promotes the development of CD4+CD25+ regulatory T cells

We have previously shown that mice lacking the IL-12-specific receptor subunit beta2 (IL-12Rbeta2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rbeta2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rbeta2(-/-) mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rbeta2-deficient mice to autoimmune diseases. T cells from IL-12Rbeta2(-/-) mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25(+)CD4(+) regulatory T cells (Tregs) in the thymus and spleen of IL-12Rbeta2(-/-) mice were comparable to those of WT mice. However, IL-12Rbeta2(-/-) mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-beta, as shown by significantly lower numbers of CD25(+)CD4(+) T cells that expressed Foxp3. Functionally, CD25(+)CD4(+) Tregs derived from IL-12Rbeta2(-/-) mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rbeta2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rbeta2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway.     

IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model.

CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-gamma released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-gamma and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.     

Pre-existing tumor-sensitized T cells are essential for eradication of established tumors by IL-12 and cyclophosphamide plus IL-12.

The antitumor immune response activated by IL-12, especially by a combination of cyclophosphamide and IL-12 (Cy+IL-12), is clinically significant in certain experimental tumor models, in that a number of well-established (10-20 mm in diameter) s.c. tumors are completely eradicated. Furthermore, Cy+IL-12 treatment is also able to eradicate well-established grossly detectable experimental lung metastases and advanced ascites tumors. Despite the dramatic antitumor effects seen in some tumor models, Cy+IL-12 fails to induce regression of other established tumors. Characterization of tumor immunogenicity shows that all tumors responding to IL-12 and Cy+IL-12 treatments are immunogenic tumors, in that an antitumor immune response is detectable in tumor-bearing hosts upon tumor establishment. In contrast, none of the nonimmunogenic tumor responds to IL-12 and Cy+IL-12 treatments. Analysis of cellular requirements for successful tumor rejection through an adoptive cell transfer approach reveals that the presence of tumor-sensitized, but not naive, T cells is essential for tumor rejection by IL-12 and Cy+IL-12. Transfer of these tumor-sensitized T cells must be conducted before, but not after, IL-12 treatment in order for tumor rejection to occur. The requirement of sensitized T cells is also tumor specific. In mice bearing immunogenic tumors, the presence of pre-existing tumor-sensitized T cells is demonstrated by adoptive cell transfer experiments using purified spleen T cells from these mice. Results from our study show that Cy+IL-12-based immunotherapy of cancer may be highly effective and that pre-existing tumor-sensitized T cells are essential for the success of the therapy.     

JNK regulates HIPK3 expression and promotes resistance to Fas-mediated apoptosis in DU 145 prostate carcinoma cells

Elevated endogenous JNK activity and resistance to Fas receptor-mediated apoptosis have recently been implicated in progression of prostate cancer and can promote resistance to apoptosis in response to chemotherapeutic drugs. In addition, JNK has been demonstrated to promote transformation of epithelial cells by increasing both proliferation and survival. Although numerous studies have reported a role for JNK in promoting Fas receptor-mediated apoptosis, there is a paucity in the literature studying the antiapoptotic function of JNK during Fas receptor-mediated apoptosis. Consequently, we have used the recently described specific JNK inhibitor SP600125 and RNA interference to inhibit endogenous JNK activity in the prostate carcinoma cell line DU 145. We demonstrated that endogenous JNK activity increased the expression of a kinase, HIPK3, that has previously been implicated in multidrug resistance in a number of tumors. HIPK3 has also been reported to phosphorylate FADD. The interaction between FADD and caspase-8 was inhibited, but abrogation of JNK activity or HIPK3 expression was found to restore this interaction and increased the sensitivity of DU 145 cells to Fas receptor-mediated apoptosis. In conclusion, we present novel evidence that JNK regulates the expression of HIPK3 in prostate cancer cells, and this contributes to increased resistance to Fas receptor-mediated apoptosis by reducing the interaction between FADD and caspase-8.